The normal ovulating woman will recruit approx. 300 immature oocytes (ova) for each menstrual cycle. Normally, during a process of apoptosis all but one oocyte will die before ovulation. Conventional in vitro fertilisation (IVF), treatment for special cases of severe male and female infertility, is based on retrieval of mature human ova followed by fertilisation of the mature oocytes with spermatozoa. The recruitment of human mature oocytes is accomplished by several complicated forms of hormone treatment regimens, often with discomfort or risk for the women involved. These hormone treatment regimens will especially become a problem in the future, as IVF is increasingly offered to perfectly normal women in these programs due to their husbands"" poor sperm quality. Furthermore, this type of treatment will normally provide a pregnancy rate of 20% per started cycle.
Because of the risk, discomfort and cost of the hormonal stimulation several other approaches have been tried during the years. In animals in vitro maturation (IVM) has become an efficient method for producing oocytes for IVF, but until now recorded success rates for clinical human IVM have been low (Cha, Trounson, Barnes, Russel). One of the most simple ways to avoid hormonal stimulation has been not to stimulate with hormones at all. This treatment regimen, however, has only provided a limited numbers of pregnancies and the calculated pregnancy rate is never beyond 5% per started cycle.
The present invention relates to the ability of a test system to predict the timing and development of specific healthy, activated prophase ova to develop further in vitro up to MF-II without exogenous hormonal treatment of the woman, and hereby avoid the degeneration of the majority of the ova, even though the woman is not treated with hormones. In contrast to all other previous hormonal assays always focusing on later stages of ova development, usually ova in the MF-II stage, the measurements aim at the timing of aspiration of non-fertilisable ova, which can mature in vitro to be fertilised. By this method pregnancy rates of more than 15% are achieved.
The nuclear development of the ova are arrested in the diplotene phase as germinal vesicles. This is referred to as the dicytate stage. This stage is characterised by highly diffuse chromosomes, the DNA of which has little affinity for such nuclear strains as Feulgen""s reagent. The chromosomes of ova in the dicytate stage bear lateral projections in the form of branches and loops which actively replicate ribonucleic acid (RNA). They closely resemble the xe2x80x9cLampbrushxe2x80x9d chromosomes, which are found almost universally in eggs of lower vertebrates and some invertebrates. The RNA may act as the messenger directing protein synthesis within the ova itself. Circumstantial evidence from studies of ova in frogs and toads suggest that some of the RNA may act as the early organiser of mammalian development. Whatever the outcome of the additional studies required to elucidate these complex problems, the dicytate stage should clearly not be referred to as a resting phase, since the oocytes show a high degree of metabolic and synthetic activity at a time when the follicular envelope consist of only a few flattened epithelia cells.
Human oocyte/folicular development is a controlled mechanism both in time and biological processes. 99% of all ova are arrested in the meiotic prophase (that is the dicytate stage) even before birth. From this pool a couple of hundred ova are selected and activated every month (the menstrual cycle) for further development that is resumption of the meiosis though germinal vesicle breakdown (GVB) and Metaphase I (MF-I) to Metaphase II (MF-II) and growth of the follicle.
The reproductive state of the human female is cyclic with a complex interaction between the hypothalamus, anterior pituitary and the ovaries eventually leading to the process of ovulation. The cycle is repeated with an average period of 28-30 days (range 25-35 days). The first phase, menstruation, last 4-5 days. The first day of a cycle is the first day of the first phase, that is the first day of menstrual bleeding. The second follicular phase of the ovary corresponds to the proliferative phase of the endometrium and lasts 10-16 days (i.e. highly variable). Then follows an ovulatory phase (36 hours) and finally a luteal phase which corresponds to the secretory phase of the endometrium and is usually constant at 14 days. Three components critical to the understanding of the menstrual cycle:
1. hypothalamic GnRH control of FSH/LH release,
2. ovarian follicular development to ovulation and subsequent corpus luteum formation and
3. the feedback control of FSH/LH secretion by ovarian hormones.
During the second half of the reproductive cycle, the corpus luteum develops and secretes both oestrogen and progesterone. Oestrogen continues to promote the proliferative activity in the endometrium. Progesterone on the other hand causes the endometrial glands to become distended with secretory products which include glycogen (important for the developing embryo should implantation occur). Endometrial blood flow increases and the spiral arteries become coiled and twisted. The second half of the cycle is called the luteal phase (ovaries) or the secretory phase (uterus). If implantation does not occur then the corpus luteum regresses, there is a rapid fall in the secretion of oestrogen and progesterone, the endometrium undergoes shrinkage due to extracellular fluid loss, the spiral arteries constrict, the endometrial blood flow decreases with cell death and destruction of blood vessels. These chances eventually lead to a phase of menstrual bleeding where all but the basal layer of the endometrium is lost. The first day of the menstrual bleeding is, for practical reasons the first day of the menstrual cyclus.
Ova maturation is the final stage of ova development that prepares for fertilisation and embryo development. It can be divided into two general processes: nuclear maturation and cytoplasmic maturation. Nuclear maturation is defined as the resumption of meiosis and progression to MF-II while cytoplasmic maturation is defined as the extragenomic changes that prepare the egg for activation, pronuclear formation, and early embryogenesis.
The follicle surrounding the ova in the dicytate stage comprises a single layer of flattened granulosa cells. The signs of further development of the ova and follicle involves further multiplication in the number of granulosa cells and also the passage of fluid into spaces between the granulosa cells. As the quantity of fluid increases, the cavities that it occupies increases in size and become confluent to form an antrum. The follicle is now said to be of the Graafian type. With the further expansion of the antrum, the ova occupies a position at one side of the follicle and is surrounded by two or more layers of granulosa cells. The innermost layer of these cells becomes columnar in the shape and constitutes the corona radiata which, as the innermost part of the cumulus oophorus, persists around the egg for a period after ovulation.
The nuclear maturation proceeds in parallel with the follicular development Thus, GVB and MF-I ova are observed during the formation of the antrum that is in the antral and pre-antral follicle. By the time the antrum is fully developed the meiotic division is completed and the first polar body extrusion is seen.
A few hundred ova reach this stage during each menstrual cycle. However, due to presently unexplained factors, the majority of these enter atresia, wherein the ova undergo an apoptotic process and die. The dissolution of the innermost part of the granulosa cells whilst the egg is still in the follicle, or precociously after its departure, is a sure sign that degenerative changes are occurring that will result in the death of the ova. Only a few, normally only 1, will proceed the development.
If the woman receives FSH, more than one follicle will proceed developmentally to MF-II avoiding atresia.
By MF-II is understood an oocyte with 1 polar body, and expanded cumulus complex and which has finally gone through a germinal vesicle break-down. These oocytes are readily recognised by a routine technician normally handling oocytes for IVF. MF-II ova are the type of ova which are prepared and able to be fertilised by the sperm cell. At no earlier stage in the development of the ova is this presently possible.
Conventional in vitro fertilisation (IVF) treatments aim at gaining as many as possible of the ova in MF-II. This has been obtained by exogenous hormonal treatment of the woman, including follicular stimulation hormone (FSH). rFSH, or urin derived FSH, is administered in the dose of 100 to 250 IU/day. Often this treatment is continued for 8-10 days. Futher treatment with GNRH agonists such as Buserelin daily are added to the treatment. By this treatment the degeneration of most of these activated ova is avoided. This type of hormonal treatment always includes the risk of hyper-stimulation syndrome and other side effects such as the increased risk of ovarian cancer, nausea, discomfort, oedema, pain, allergic reaction to the medication, depression and weight gain, all of which are hard to justify specifically if the woman is healthy and the reason for the IVF treatment is due to a severe male infertility problem. Thus, at the moment, more than 30% of all IVF cycles in the world are initiated with heavy medication of the women although the problem is a male factor.
In order to obtain healthy MF-II ova, the hormonal treatment of the woman has been supplemented with monitoration of serum hormone levels of Estradiol, FSH, LH, progesterone, PP14, and/or PP12. The timing of the aspiration of healthy MF-II ova has further been aided by ultrasound scanning of the follicular development. All of these clinical tests have been devised to increase the healthiness and number of recruitable MF-II ova.
IVM preferably starts with immature or not fully matured gametes. In the woman the ova will be recognised as oocytes with a tight cumulus mass, no polar bodies or Germinal vesicles visible. These ova are readily recognised by a person involved in routine IVF-treatments as being immature ova.
If no hormones are administrated only one oocyte will mature as seen in normal ovulating women. By releasing the immature oocytes from the ovary prior to initiation of the apoptotic processes wherein the ova enter atresia, and subsequently mature the ova further in vitro, the women can avoid the risk and discomfort associated with hormonal treatment and still a sufficient number of mature Metaphase II (MF-II) ova will be available for subsequent in vitro fertilisation.
It has been possible to produce oocytes whose nuclear maturation has progressed to MF-II, but which are incompetent to complete preimplantation development. The importance of cytoplasmic control over developmental competence has been described in the immature monkey oocyte. Using micromanipulation, ooplasm was removed from MF-II oocytes and injected into prophase I oocytes. Monkeys receiving the oocytes with cytoplasmic transfusion had a sevenfold increase in pregnancy rate compared to oocytes without ooplasm injection (Flood).
Up to this date IVM protocols published have either used a fixed day in the menstrual cycle, calculated from the previous ovulation or the onset of menses, or just used a convenient day prior to final development of the antral follicles. This has gained pregnancy rates from 0%-10%.
The present invention relates to the finding that the measurement of specific signalling hormones will be indicative of the appropriate timing of ova aspiration for an IVM procedure. This measurement being indicative of the timing of aspiration of ova will optimise the number of ova specifically capable of further maturation in vitro, fertilisation and further development. The identification of specific signalling hormones as predictive markers for when non-fertilisable ova capable of in vitro maturation and subsequent fertilisation, cleavage, implantation and pregnancy are present in the mammal greatly increases the rate of success in IVM cycles.
A non-fertilisable ova is in this patent application an immature ova (=oocyte) that upon contact with a mature sperm cell will not complete the meiotic division and accept the genetic material from the sperm cell and form a fertilised cell. It is presently anticipated that the endocrine and intra-ovarian regulation of ova maturation will leave the ova with a precise, but complex composition of substances with specific concentrations or concentration ratios, such that the non-fertilisable ova after in vitro maturation to MF-II will be capable of fertilisation, subsequent cleavage and then pregnancy after implantation into the female mammal.
One aspect of the invention thus relates to an assay for the presence or quantity of at least one predictive marker in a sample from a mammal wherein a specific determination of the presence or quantity of the at least one predictive marker indicates when non-fertilisable ova capable of in vitro maturation and subsequent fertilisation are present in the mammal.
The assay for the at least one predictive marker is based on a sample from a female mammal. In one embodiment of the invention the sample is a sample selected from the group consisting of e.g. Body secrete, body fluid, cellular nutrients, follicle content, sputum, blood, urine, faeces, uterus or vaginal secretes and components, menstruation products, epitelia and epitelia derived components, skin components, and dead or living cells. A person skilled in the art will readily understand how each of these samples are obtained.
In one embodiment of the present invention, the sample originates from a mammal, such as a pet, e.g. cat, dog, or guinea pig; or a zoo animal e.g. a primate. In another embodiment of the invention, the mammal is a laboratory animals such as a rat, or a mouse. In further preferred embodiments, the mammal is part of the industry, preferably a farm animal such as cattle, a horse, pig, mink, goat, or sheep. In the presently most preferred embodiment, the mammal is a human being.
In one aspect of the invention, the female mammal is tested prior to ova aspiration. Possibly more than one test is performed. Either an absolute cut off level or a relative measure such as a plateau, an increase or a decrease of the before mentioned selective marker can predict the timing of the ova aspiration for IVM.
In this context, a predictive marker should be understood as any substance that will change concentration, colour, structure, confirmation, and/or reaction pattern in a biological, physical or chemical test. It is to be understood that a lot of potential predictive markers exist. In one embodiment of the invention the assay is an assay, wherein the at least one predictive marker is a hormone selected from the group consisting of gonadotrophins (such as FSH, LH, prolactin, and HCG), thyoidea hormones (such as TSH, T3, and T4), and steroid hormones (such as sex hormones: Estradiol, estrogens, estratriol, progesterone, and testosterone; androgenes: cortisone or cortisole). In another embodiment of the invention the assay is an assay, wherein the at least one predictive marker is a small peptide hormone (such as GIP or VIP). In another embodiment of the invention the assay is an assay, wherein the at least one predictive marker is a peptide hormone (such as PP12, PP14, an inhibin: inhibin A or inhibin B: activin, or xcex11 or xcex12 globulins). In another embodiment of the invention the assay is an assay, wherein the at least one predictive marker is a lipid selected from the group consisting of Meiosis Activating Sterols (MAS) lipids and prostaglandines. In another embodiment of the invention, the assay is an assay, wherein the at least one predictive marker is a nucleic acid selected from the group consisting of DNA fragments, mRNA, and tRNA. In another embodiment of the invention the assay is an assay, wherein the at least one predictive marker is a marker selected from the group consisting of LFN1, LFN2, LFN3, glycoconjugates, carbohydrates, cellular nutrients, integrins such as integrin 1, and carbohydrate epitopes. In another embodiment of the invention the assay is an assay, wherein the at least one predictive marker is an intra- or intercellular messenger such as cAMP. In yet another embodiment of the present invention the assay is an assay wherein the at least one predictive marker is an enzyme, such as phosphodiesterase or an inhibitor of phosphodiesterase.
Due to the high complexity of the interaction between and in-between proteins, hormones, lipids, nucleic acids, intercellular messengers, and enzymes, it is presently anticipated that the timing of the aspiration of non-fertilisable ova shall be dependent on combinations of cut off levels of certain predictive markers and/or relative measurements on one or more predictive markers, i.e. relative measurements of the same marker where the samples have been taken at different times, or relative measurements of two or more markers present in a sample at the same time.
The assay comprises at least one predictive marker. That is two predictive markers such as three, four, five or six predictive markers.
It is more than likely that the predictive value of the assay will be markedly increase if the evaluation of two or more predictive markers is combined.
Based on the description in examples 1 and 2 on how the correlation between the two predictive markers Inhibin A and Estradiol correlate to the outcome of the IVM cycle, the person skilled in the art will be able to determine such correlations between other predictive markers. In one embodiment, there is a significant correlation between the chance of pregnancy in that cycle and the relative increase, or relative decrease, in the concentration of the predictive marker subject to investigation in the sample from day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, or even day 9 prior to the day of ova aspiration. In another embodiment, there is a significant correlation between the chance of pregnancy in that cycle and the absolute increase, the absolute decrease, or the plateau in the concentration of the predictive marker subject to investigation in the sample from day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, or even day 9 prior to the day of ova aspiration. In yet another embodiment, there is a significant correlation between the concentration of the predictive marker subject to investigation in the sample on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, or even day 9 and the chance of pregnancy in that cycle. In one aspect of this embodiment, the significant correlation between the concentration of the predictive marker subject to investigation in the sample and the chance of pregnancy in that cycle is reduced to a binary correlation, such that a cut-off level is set, and the chance of pregnancy in that cycle is correlated to whether the concentration of the predictive marker subject to investigation is more or less than the cut-off level.
In one embodiment of the invention the pregnancy rate in a study of IVM-cycles wherein the time for ova aspiration has been set by an assay as described in the present invention, is more than 10%, such as more than 13%, 15%, 18%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35% or even more than 40%.
In a presently preferred embodiment of the present invention two predictive markers are chosen. That is Inhibin A and Estradiol. As illustrated in examples 1 and 2 an increase in Estradiol and Inhibin A predicts success of the IVM-cycle.
It will sometimes be observed that the increase in Estradiol is seen prior to the increase in Inhibin A. In that case, the concentration of Estradiol might fall, or reach a plateau, by the time the increase in Inhibin A is observed. In this case it is still considered that an increase in Estradiol has been reached such that the chance for success is high, due to the fact that the increase in Inhibin A is the major factor. This is especially the case, when FSH priming is performed (see example 3).
In a presently most preferred embodiment of the present invention, the assay comprises three specific reactions and results for two selective markers. The first specific reaction is a measurement of the concentration of Inhibin A of less than 10 pg/ml in a blood sample on day 3 of the menstrual cycle. The second specific reaction and result is an increase in the concentration of Inhibin A of more than 80% from day 3 to the day of ova aspiration. The third specific reaction and result is an increase in the concentration of Estradiol of more than 87% from day 3 to the day of ova aspiration. Thus in one aspect of the embodiment, the woman will have a blood test drawn on day 3 of the menstrual cycle and the concentrations of Inhibin A and Estradiol in the blood sample are determined. If the concentration of Inhibin A is less than 10 pg/ml in said blood sample, this cycle will not be an IVM-cycle. However, if the concentration of Inhibin A is more than or equal to 10 pg/ml in said blood sample a new blood sample will be withdrawn on day 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16 unless the second specific and the third specific reaction have been observed. If the second specific and/or the third specific reaction are observed, the day of the observation should be the day, where ova are aspirated. It is contemplated, based on the results from example 2, that IVM cycles wherein the time for ova aspiration have met these three specific reactions will have pregnancy rates of more than 12%, such as more than 16%, 22%, 25%, 26% or even more than 28%.
As will be appreciated by the person skilled in the art, measurements of predictive markers in mammalian samples are routine measures. In one aspect of the invention, the assay is prepared in such a way that the specific reaction can be compared and compiled without the assistance of doctors or advanced medical equipment. However, as will be understood by the person skilled in the art, quite a few of the predictive markers can presently not be measured without expensive equipment making it necessary to transfer the sample to a laboratory setting, where the result of the assay will guide the timing of ova aspiration in the mammal.
Due to the complexity of the concentrations, relative increases, relative decreases, absolute increases, absolute decreases, plateau""s and cut off values, one aspect of the invention is a scheme to be filled out during the cause of the menstrual cycle wherein IVM-treatment is contemplated. In one embodiment the scheme comprises columns for each day in the menstrual cycle and rows for each of the predictive markers such that each cell can be filled out as the measurements of the predictive markers are obtained. Automated, semi-automated, and guided manual calculations of the alterations in the predictive markers will ease interpreting the results of the specific reactions.
In one embodiment of the invention, the assay for predictive markers is used as a first selection criteria. If this criteria is fulfilled the woman will be subjected to ultrasonic measurements of the uterus and ovaries. If the evaluation of the ultrasonic measurements determines:
a large number of antral follicles,
the size of the leading follicle is about 10 mm,
the cohort of follicles is uniform,
the endometrial thickness is more than about 3 mm, and
beginning trilaminaer structures of the endometrial lining
the likelihood of having found the right time for successful ova aspiration, in vitro maturation to MF-II, fertilisation, implantation and pregnancy is very high.
In a presently preferred embodiment of the present invention, the ova is derived from a female human being having an ovarian follicle with a diameter of 8-12 mm. The advantage of such small follicles is that they are present in substantial numbers at this time in the menstrual cycle without prior severe hormonally treatment, they can bee seen by ultrasound, and an ultrasonically guided transvaginal puncture of the follicles is possible to perform in order to retrieve the oocytes.
It will be understood by the person skilled in the art that other sizes of ova should be aspirated from other mammals.
In one embodiment of the invention, the general knowledge of the women""s menstrual cycle is incorporated in the timing of the aspiration of ova. Thus, usually the aspiration is performed between day 6 and 17, that is usually between day 9 and 11.
Thus, in one embodiment of the present invention, a method to determine the timing of ova aspiration comprises the following steps:
a) assay at least one predictive marker in a sample from a mammal;
by the time the result of the specific reaction in the assay in step a) predicts the presence of non-fertilisable ova capable of in vitro maturation and subsequent fertilisation in the mammal
b) evaluate the time relative to the first day of the menstrual bleeding;
if day is between day 6 and day 17 of the menstrual cycle then
c) evaluate an ultrasound picture of the ovaries of the mammal;
if the size of the follicles is between 8 mm and 12 mm and the cohort of follicles is uniform, then
d) evaluate an ultrasound picture of the uterus of the mammal;
if the endometrial thickness is more than about 3 mm and beginning trilaminaer structures of the endometrial lining are observed, the time for ova aspiration is right. However, if any of the above mentioned steps fail, the cycle is probably not suitable for IVM, and the aspiration of ova should be postponed until next cycle, where the method is repeated.
The ability of IVM oocytes to complete meiosis and to become developmentally competent may be enhanced by FSH priming prior to oocyte aspiration. In human IVM a short FSH stimulation doubles the number of oocytes recovered per patient as well as increases the proportion of oocytes that attain MF-II after 48 hours. In one embodiment of the present method, the woman is treated on days 3, and/or 4, and/or 5 in the menstrual cycle with hormones such as FSH followed by a discontinuation of the treatment. In example 3, an increased maturation rate and cleavage rate was observed in the FSH priming group with a delay between the last injection and the aspiration compared to the FSH priming without a delay. In the FSH priming group with a delay between the last injection and the aspiration, an increased maturation rate was observed compared to unstimulated oocytes, but the cleavage rate of the matured MF-II oocytes was not affected. It was high in both groups. An early apoptotic phase or an artificial plateau phase in the follicular growth may mimic the final preovulatory follicular maturation in terms of developmental competence. In this way an artificial plateau or fall of the Estradiol level is obtained causing an apoptotic process in the ovary leading to the above mentioned signalling into the immature oocyte. Such a fall in Estradiol level is expected around day 10 in the menstruation cycle. Retrieval of oocytes in women without hormonal stimulation should preferably be done on day 10 in the menstruation cycle, as that day statistically is associated with numerous oocytes in a prophase.
In one aspect of the invention the woman will benefit from an initial treatment on days 3, 4, and 5 in the menstrual cycle with a hormone such as FSH followed by a discontinuation of treatment. In this treatment regimen, the blood level of Estradiol in the woman might be monitored with the object of selecting maturating oocytes, indirectly measured by none growing (growth pause) follicles. Early apoptotic oocytes destined for becoming apoptotic are characterised in that they are easily detached from the ovary during the puncture of the follicles and they have a compact cumulus mass. Then by the time of a plateau or fall in Estradiol level is observed, the retrieval of oocytes is performed. An observed plateau or fall in Estradiol concentration is due to a prior increase in the Estradiol concentration between day 3 of the menstrual cycle and the day of ova aspiration. In one embodiment the time for ova aspiration is the time of an increase in Estradiol concentraton, that is before the plateau or fall in Estradiol concentration. In a further preferred embodiment, the monitoration of Estradiol concentration is further supplemented with a monitoration of the Inhibin A concentration.
Thus, one embodiment of the present invention is a kit comprising the suitable FSH medication and an assay as described above. Optionally, the kit further comprises means to obtain the sample or samples for the assay.
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